Use of 1-(β-D-arabinofuranosyl) -5-propynyluracil for lowering serum cholesterol

ABSTRACT

The use of 1-(β-D-arabinofuranosyl)-5-propynyluracil or a pharmaceutically acceptable salt or physiologically labile ester thereof for lowering serum cholesterol, particularly low density lipoprotein cholesterol levels in a human subject.

The present invention relates to a new therapeutic use for a 5-substituted pyrimidine nucleoside.

1-(β-D-arabinofuranosyl)-5-propynyluracil is already known as an anti-viral agent having activity against viruses of the herpes group, in particular Varicella Zoster virus and cytomegalovirus. In this connection reference can be made to EP-A-0 417 560.

The role of elevated serum cholesterol levels in many pathological conditions in man is now well established and deposits of cholesterol in the vascular system have been implicated as causative of a number of pathological conditions such as coronary heart disease. Cholesterol is found in the blood in the form of complex particles which generally consist of a core of cholesterol esters and triglyceride surrounded by phospholipid and protein. Cholesterol is carried to sites where it is deposited in blood vessels in the form of so-called low density lipoprotein (LDL) cholesterol and is removed from such sites in the form of so-called high density lipoprotein (HDL) cholesterol.

It has now surprisingly been found that 1(β-D-arabinofuranosyl)-5-propynyluracil is effective in lowering serum cholesterol, and particularly LDL cholesterol, levels in cynomolgus monkeys and preliminary studies indicate a similar effect in human subjects.

Accordingly, the present invention provides a method for lowering serum cholesterol, particularly LDL cholesterol, levels in a human subject which comprises administering to the said subject an effective amount of 1-(β-D-arabinofuranosyl)-5-propynyluracil or a pharmaceutically acceptable salt or physiologically labile ester thereof.

Examples of clinical conditions which may be treated in accordance with the present invention include polygenic hypercholesterolaemia, familial hypercholesterolaemia, LDL receptor deficiency/abnormality, apoB deficiency/abnormality, familial combined hyperlipidaemia, apoE2 homozygosity, apoC2 deficiency and familial lipoprotein lipase deficiency.

The present invention also provides the use of 1(β-D-arabinofuranosyl) -5-propynyluracil or a pharmaceutically acceptable salt or physiologically labile ester thereof for the manufacture of a medicament for lowering serum cholesterol, particularly LDL cholesterol, levels in a human subject.

The present invention also provides a pharmaceutical composition for lowering serum cholesterol, particularly LDL cholesterol, levels in a human subject, which comprises 1(β-D-arabinofuranosyl)-5-propynyluracil or a pharmaceutically acceptable salt or physiologically labile ester thereof, together with at least one pharmaceutically acceptable carrier or diluent.

1(β-D-arabinofuranosyl)-5-propynyluracil, pharmaceutically acceptable salts and physiologically labile esters thereof, are known compounds and may be prepared by the methods described in EP-A-0 417,560. Suitable pharmaceutically acceptable salts for use according to the invention include salts formed with physiologically acceptable bases, for example alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and NX₄ ⁺ (where X is for example C₁₋₄ alkyl) salts. Pharmaceutically acceptable salts of 1(β-D-arabinofuranosyl) -5-propynyluracil may be prepared in conventional manner, for example by reaction of 1(β-D-arabinofuranosyl)-5-propynyluracil with the appropriate base.

As used herein, the term "physiologically labile ester" refers to an ester of 1-(β-D-arabinofuranosyl)-5-propynyluracil which upon administration to a human subject is converted to 1(β-D-arabinofuranosyl) -5-propynyluracil or a metabolite thereof having the same or similar cholesterol lowering activity to the parent compound. Examples of suitable mono- and di-esters include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain alkyl, alkoxyalkyl, carboxyalkyl, aralkyl, aryloxyalkyl and aryl (including aryl substituted by a substituent such as halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy); sulphonate esters such as alkyl- and arylalkylsulphonyl; mono- di- and tri-phosphate esters, which may or may not be blocked; amino acid esters; and nitrate esters. Any reference to an ester also includes that ester in the form of a pharmaceutically acceptable salt.

1-(β-D-arabinofuranosyl)-5-propynyluracil or a pharmaceutically acceptable salt of physiologically labile ester thereof (referred to hereinafter as the "active ingredient"), may be administered by any route appropriate to the condition of the subject, suitable routes including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous. intradermal, intrathecal and epidural). It will be appreciated that the preferred route of administration may vary, for example, with the condition of the subject.

The amount of the active ingredient required for administration to a human subject will depend upon a number of factors including the condition and identity of the subject and will ultimately be at the discretion of the attendant physician. In general, a suitable, effective dose will be in the range 20 mg to 1 g per day, preferably 50 to 800 mg per day, most preferably 100 to 400 mg per day. Unless otherwise stated, all weights of the active ingredient are calculated as the weight of the parent compound and the weights would need to be increased proportionately for salts and esters.

The necessary dose of active ingredient is preferably administered as a single unit dose once daily, but may, if desired, be presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms containing, for example, 20 to 500 mg, preferably 100 to 400 mg, of active ingredient per unit dosage form.

While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention comprise the active ingredient, together with one or more pharmaceutically acceptable carriers or diluents and optionally therapeutic ingredients. The carrier(s) or diluents must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof.

The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant. inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-liked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspension may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The invention is illustrated by the following Examples of Pharmaceutical Compositions and Biological Data.

EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

The following examples illustration pharmaceutical formulations according to the invention in which the active ingredient is 1-(β-D-arabinofuranosyl)-5-propynyluracil.

Example 1

    ______________________________________                                         Tablet                                                                         ______________________________________                                         Active ingredient      100    mg                                               Lactose                200    mg                                               Starch                 50     mg                                               Polyvinylpyrrolidone   5      mg                                               Magnesium Stearate     4      mg                                                                      359    mg                                               ______________________________________                                    

Tablets are prepared from the forgoing ingredients by wet granulation followed by compression.

Example 2

Tablet Formulations

The following formulations A, B and C are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

    ______________________________________                                                         mg/tablet                                                                              mg/tablet                                              ______________________________________                                         Formulation A                                                                  Active ingredient 250       250                                                Lactose B.P.      210        26                                                Povidone B.P.      15        9                                                 Sodium Starch Glycollate                                                                          20        12                                                Magnesium Stearate                                                                                5         3                                                                   500       300                                                Formulation B                                                                  Active ingredient 250       250                                                Lactose           150       --                                                 Avicel PH 101 ®                                                                               60        26                                                Povidone B.P.      15        9                                                 Sodium Starch Glycollate                                                                          20        12                                                Magnesium Stearate                                                                                5         3                                                                   500       300                                                ______________________________________                                         Formulation C                                                                                    mg/capsule                                                   ______________________________________                                         Active Ingredient 100                                                          Lactose           200                                                          Starch             50                                                          Povidone           5                                                           Magnesium Stearate                                                                                4                                                                             359                                                          ______________________________________                                    

The following formulations, D and E, are prepared by direct compression of the fixed ingredients. The lactose used in formulation E is of the direct compression type.

    ______________________________________                                                           mg/capsule                                                   ______________________________________                                         Formulation D                                                                  Active ingredient   250                                                        Pregelatinised Starch NF15                                                                         150                                                                            400                                                        Formulation E                                                                  Active ingredient   250                                                        Lactose             150                                                        Avicel ®        100                                                                            500                                                        ______________________________________                                    

The following formulation F is prepared by wet granulation of the ingredients with a solution of povidone followed by the addition of magnesium stearate and compression.

    ______________________________________                                         Formulation F                                                                                    mg/tablet                                                    ______________________________________                                         Active ingredient   500                                                        Hydroxypropylmethylcellulose                                                                       112                                                        (Methocel K4M Premium) ®                                                   Lactose B.P.         53                                                        Povidone B.P.C.      28                                                        Magnesium Stearate   7                                                                             700                                                        ______________________________________                                    

Drug release takes place over a period of about 6-8 hours and is complete after 11 hours.

Example 3

A capsule formulation A is prepared by admixing the ingredients of Formulation D in Example 2 above and filling into a two-part hard gelatin capsule. Formulation B as follows is prepared in a similar manner.

    ______________________________________                                         Formulation B                                                                                   mg/capsule                                                    ______________________________________                                         Active ingredient  250                                                         Lactose B.P.       143                                                         Sodium Starch Glycollate                                                                           25                                                         Magnesium Stearate  2                                                                             420                                                         ______________________________________                                    

A further formulation C is prepared as follows:

    ______________________________________                                         Formulation C                                                                                 mg/capsule                                                      ______________________________________                                         Active Ingredient                                                                               250                                                           Macrogel 4000 BP 350                                                                            600                                                           ______________________________________                                    

Capsules are prepared by melting the Macrogel 4000 BP, dispersing the active ingredient in the melt, and filling the melt into a two-part hard gelatin capsule.

The following controlled release capsule formulation D is prepared by extruding the first three ingredients using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with a release-controlling membrane of ethyl cellulose and filled into a two-piece hard gelatin capsule.

    ______________________________________                                         Formulation D                                                                                   mg/capsule                                                    ______________________________________                                         Active Ingredient  250                                                         Microcrystalline Cellulose                                                                        125                                                         Lactose BP         125                                                         Ethyl Cellulose     13                                                                            513                                                         ______________________________________                                    

Example 4

    ______________________________________                                         Injectable Formulation                                                         ______________________________________                                         Active ingredient       0.200 g                                                Sterile, pyrogen free     10 ml                                                phosphate buffer (pH 7.0) to                                                   ______________________________________                                    

The active ingredient is dissolved in most of the phosphate buffer (35°-40° C.), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Example 5

    ______________________________________                                         Intramuscular Injection                                                        ______________________________________                                         Active ingredient       0.20 g                                                 Benzyl Alcohol          0.10 g                                                 Glycofurol 75           1.45 g                                                 Water for Injection q.s. to                                                                            3.00 ml                                                ______________________________________                                    

The active ingredient is dissolved in the glycofural. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Example 6

    ______________________________________                                         Syrup Suspension                                                               ______________________________________                                         Active ingredient       0.2500 g                                               Sorbitol Solution       1.5000 g                                               Glycerol                2.0000 g                                               Dispersible Cellulose   0.0750 g                                               Sodium Benzoate         0.0050 g                                               Flavour, Peach 17.42.3169                                                                              0.0125 ml                                              Purified Water q.s. to  5.0000 ml                                              ______________________________________                                    

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dissolved. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

Example 7

    ______________________________________                                         Suppository                                                                                       mg/suppository                                              ______________________________________                                         Active ingredient (63 μm)*                                                                        250                                                      Hard Fat, BP         1770                                                      (Witepsol H15 - Dynamit Nobel) ®                                                                2020                                                      ______________________________________                                          *The active ingredient is used as a powder wherein at least 90% of the         particles are of 63 μm diameter or less.                              

One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45° C. maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45° C., the remaining Witepsol H15 is added to the suspension which is stirred to ensure a homogenous mix. The entire suspension is then passed through a 250 μm stainless steel screen and, with continuous stirring, allowed to cool to 40° C. At a temperature of 38°-40° C., 2.02 g aliquots of the mixture are filled into suitable plastic moulds and the suppositories allowed to cool to room temperature.

Example 8

    ______________________________________                                         Pessaries                                                                                      mg/pessary                                                     ______________________________________                                         Active ingredient 63 μm                                                                       250                                                          Anhydrate Dextrose                                                                               380                                                          Potato Starch     363                                                          Magnesium Stearate                                                                                7                                                                             1000                                                         ______________________________________                                    

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

BIOLOGICAL DATA

A. Cynomolgus Monkeys

The effect of 1(β-D-arabinofuranosyl)-5-propynyluracil in the cynomolgus monkey may be regarded as predictive of its effect in man. 1(β-D-arabinofuranosyl)-5-propynyluracil was administered by gavage to groups of three male (sexually mature) and three female cynomolgus monkeys, twice daily (6 hour interval between doses) to give total doses of 0, 25, 50, 100 and 500 mg/kg/day for 28 days. An additional two male and two female animals were dosed at 0 and 500 mg/kg/day and were then allowed a two week treatment-free period to assess reversibility. Blood samples were obtained at selected intervals at the beginning and end of the study for biochemical analysis.

Total cholesterol levels were consistently reduced to 70% of pre-dose values from Day 3 in animals dosed at 100 and 500 mg/kg/day and from Day 7 in animals dosed at 25 and 50 mg/kg/day. Cholesterol levels remained lowered at all dose levels until the end of the study, but were restored to pre-dose values 7 days after completion of treatment.

B. Human Volunteers

Three groups of six young human volunteers were subjected to a cross over study in which each group received either 25 mg, 50 mg or 100 mg of 1(β-D-arabinofuranosyl)-5-propynyluracil twice daily for 10 days and an identical placebo for the same period. The results showed a downward trend in mean plasma cholesterol concentration as follows:

    ______________________________________                                                    Mean Plasma Cholesterol Level                                       Dosage Regimen                                                                              Before Treatment                                                                             After Treatment                                     ______________________________________                                          25 mg twice daily                                                                          5.0 mmol/l    4.9 mmol/l                                           50 mg twice daily                                                                          4.9 mmol/l    4.3 mmol/l                                          100 mg twice daily                                                                          5.1 mmol/l    4.7 mmol/l                                          ______________________________________                                    

The effects appeared to be predominantly on the LDL cholesterol fraction and there was no change in HDL cholesterol. The results provide no evidence that the maximum response has been achieved although measurement of plasma concentration of 1-(β-D-arabinofuranosyl) -5-propynyluracil suggested that the higher the peak plasma concentration of 1(β-D-arabinofuranosyl)-5-propynyluracil, the greater the fall in plasma cholesterol level. No effect was noted on plasma triglyceride levels. 

We claim:
 1. A method of lowering an elevated serum cholesterol level in a human having said elevated serum cholesterol level which comprises administering to said human an effective elevated serum cholesterol lowering amount of the compound 1(β-D-arabinofuranosyl) -5-propynyluracil or a pharmaceutically acceptable salt or labile ester thereof.
 2. A method of lowering an elevated serum cholesterol level in a monkey having said elevated serum cholesterol level comprising administering to said monkey an effective elevated serum cholesterol lowering amount of the compound 1-(β-D-arabinofuranosyl) -5-propynyluracil or a pharmaceutically acceptable salt or labile ester thereof. 